Summary The degeneracy of the genetic code is implied in the need for 61 sense codons to specify 20 different amino acids and, with the exception of methionine and tryptophan, each amino acid is encoded by more than one codon. This discrepancy between codon and amino acid numbers was first explained by Crick's wobble hypothesis, which invoked flexibility between the first anticodon and third codon positions during decoding. Since the inception of the wobble rules, over 100 posttranscriptional modifications have been described with the largest number affecting the anticodon of tRNA. As anticodon modifications accrue, new findings lead to a constant reinterpretation of the wobble rules to include novel effects on tRNA function. Anticodon-sequence alterations that expand decoding capacity are part of a growing number of post-transcriptional changes collectively known as tRNA editing. It is our view that tRNA editing provides a mechanism to effectively accommodate genetic code degeneracy. Editing can also be utilized to regulate gene expression. Furthermore, editing itself can be influenced by the structural context of an editing site and in the case of tRNA can be modulated by posttranscriptional modifications. In this proposal, we have focused on the process of inosine formation in the tRNAs of trypanosomatids. We have discovered the first example of two editing events in a single tRNA, whereby positions 32 and 34 of tRNAThr undergo C to U and A to I editing respectively. The finding that every inosine containing tRNA also undergoes C to U editing at position 32 (5'of the wobble position) raises important questions as to what role the two editing events play in the function of this tRNA. By establishing an in vitro A to I editing assay, we have demonstrated that C to U stimulates A to I editing in vitro. We have also identified the enzyme responsible for A to I editing and shown that a unique feature of this enzyme is its ability to perform two different deamination reactions and also is able to utilize both DNA and RNA as substrates. This proposal will thus focus on answering the specific questions of the nature of the machinery that specifies A to I editing and define how these enzymes achieve their specificity. As an essential step in tRNA maturation in trypanosomatids (Leishmania and Trypanosoma), these types of editing also provide a very attractive target for therapeutic intervention against parasites of very major medical importance. Given the link between tRNA maturation and disease, these studies will further expand our knowledge of the role tRNA processing plays in cellular function. PUBLIC HEALTH RELEVANCE: Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, tRNAs undergo post-transcriptional editing changes that are unique to this system. The enzyme responsible for tRNA editing changes in trypanosomatids possesses substrate specificities that are not shared with any other member of this family of proteins. It is thus important to define what features of these enzymes give them their unique specificity. The propose studies will determine the basis for substrate discrimination of the T. brucei editing enzyme which in the future may open doors towards the development of drugs against this essential activity. These studies will also provide functional and evolutionary insights into important members of the cytidine deaminase (CDA) superfamily.